A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF). One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users. Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases. Identification of protiens that may be involved in DNA replication and repair of chromatin. Identification of binding partners and PTMs of metal responsive transcription factor 1. Identification of PTMs of CD34. Identification of binding partners and PTMs of KCNH2, TRbeta, and slowpoke. The core also is performing value added research in separations (off-gel electrophoresis) and affinity techniques (IMAC and MOAC)to aid in protein and PTM identifications. Other published projects or projects with manuscriptis in preparation include: Stim1 Phosphorylation: Store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ currents (Icrac) are strongly suppressed during mitosis, the only known physiological situation in which Ca2+ store depletion is uncoupled from the activation of Ca2+ influx. We found that the ER Ca2+ sensor, STIM1, failed to rearrange into near-plasma membrane puncta in mitotic cells, a critical step in the SOCE activation pathway. We also found that STIM1 from mitotic cells is recognized by the phosphospecific MPM-2 antibody, suggesting that STIM1 is phosphorylated during mitosis. Removal of 10 MPM-2-recognition sites by truncation at amino acid 482 abolished MPM-2 recognition of mitotic STIM1, and significantly rescued STIM1 rearrangement and SOCE responses in mitosis. We identified S486 and S668 as mitosis-specific phosphorylation sites, and STIM1 containing mutations of these sites to alanine also significantly rescued mitotic SOCE. Therefore, phosphorylation of STIM1 at S486 and S668, and possibly other sites, underlies suppression of SOCE during mitosis. Phosphorylation of SIRT1: SIRT1, an NAD+-dependent protein deacetylase, plays an important role in a variety of physiological processes including stress response and energy metabolism. We have demonstrated that SIRT1 and anti-apoptotic members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family, DYRK1A and DYRK3, directly phosphorylate SIRT1 at the Threonine 522 residue. DYRK-mediated phosphorylation of Thr522 increases SIRT1 activity, resulting in deacetylation and inhibition of p53 upon DNA damage. Phosphorylation of TTP in the presence of CIN85: Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that is considered to be an anti-inflammatory protein. We found that co-expression of CIN85 with hTTP resulted in the increased phosphorylation of hTTP at serine residues in positions 66 and 93, possibly due in part to the demonstrated association of mitogen-activated protein kinase kinase kinase 4 (MEKK4) to both proteins. These data demonstrate that CIN85 binding to human TTP leads to increased phosphorylation of hTTP, possibly through association with MEKK4, with potential but unknown functional consequences. Identification and Characterization of AChBP: We identified a homolog of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella capitata, from the annelid phylum. Mass spectrometry results indicate that Asn122 and Asn216 of cc-AChBP are glycosylated when expressed using HEK293 cells. Small angle X-ray scattering (SAXS) data suggest that the overall shape of cc-AChBP is similar to homologs with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and bungarotoxin bind to cc-AChBP with high affinity, and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca. Phosphorylation of the Glucocorticoid Receptor: We discovered a new target for GSK-3beta phosphorylation: the human glucocorticoid receptor (GR). Glucocorticoid signaling is essential for life and regulates diverse biological functions from cell growth to metabolism to apoptosis. Specifically, we found hormone-dependent GR phosphorylation on serine 404 by GSK-3beta. Cells expressing a GR that is incapable of GSK-3beta phosphorylation had a redirection of the global transcriptional response to hormone, including the activation of additional signaling pathways, in part due to the altered ability of unphosphorylatable GR to recruit transcriptional cofactors CBP/p300 and the p65 (RelA) subunit of NF-kappaB. Collectively, our results describe a novel convergence point of the GSK-3beta and the GR pathways, resulting in altered hormone-regulated signaling. Our results also provide a mechanism by which GSK-3beta activity can dictate how cells will ultimately respond to glucocorticoids. Aminohydantoin lesions in DNA: The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-chloroperbenzoic acid (m-CPBA) and dimethyldioxirane (DMDO) as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d(TTGTT).(AACAA) with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification, suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism has been probed by oxidation of the monomeric species dGuo, dGMP, and dGTP. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker. Additional projects that have required more than negligible resources include efforts performed with the Akiyama, Archer, Birnbaumer, Eddy, Garantziotis, Hall, and Negishi laboratories.